Anti-histone therapy for vascular necrosis in severe glomerulonephritis

ABSTRACT

Severe glomerulonephritis involves cell necrosis as well as NETosis, programmed neutrophil death leading to expulsion of nuclear chromatin and neutrophil extracellular traps (NETs). Histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells. This was prevented by histone-neutralizing agents anti-histone IgG, activated protein C and heparin. Histone toxicity on glomeruli was TLR2/4-dependent. Anti-GBM glomerulonephritis involved NET formation and vascular necrosis. Pre-emptive anti-histone IgG administration significantly reduced all aspects of glomerulonephritis, including vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment and activation of glomerular leukocytes and glomerular crescent formation. Subjects with established glomerulonephritis treated with anti-histone IgG, recombinant activated protein C, or heparin all abrogated severe glomerulonephritis suggesting that histone-mediated glomerular pathology is a subsequent, not initial event in necrotizing glomerulonephritis. Neutralizing extracellular histones is therapeutic in severe experimental glomerulonephritis.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/746,997, filed Jun. 23, 2015, which claimed the benefit under 35U.S.C. 119(e) of provisional U.S. patent application Ser. No.62/016,277, filed Jun. 24, 2014.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 29, 2015 isnamed IMM346US1_SL.txt and is 53,943 bytes in size.

FIELD OF THE INVENTION

The invention relates to compositions and methods of use ofhistone-neutralizing agents, such as anti-histone IgG, activated proteinC, or heparin, for treatment of vascular necrosis in severeglomerulonephritis. In certain preferred embodiments, thehistone-neutralizing agent is an anti-histone antibody orantigen-binding fragment thereof, such as the BWA-3 anti-H4 antibody. Inother embodiments, the anti-histone antibodies bind to human histonesH2B, H3 or H4. More particular embodiments may concern chimeric or morepreferably humanized forms of anti-histone antibodies. However, anyother known histone-neutralizing agent may be utilized for treatingvascular necrosis in severe glomerulonephritis.

BACKGROUND

Rapidly progressive glomerulonephritis (RPGN) is a kidney syndromecharacterized by rapid loss of renal function. If untreated RPGN canresult in acute renal failure and death within months. In about 50% ofcases, RPGN is associated with an underlying disease, such asGoodpasture syndrome, systemic lupus erythematosus, or granulomatosiswith polyangitis; the remaining cases are idiopathic. RPGN encompasses aheterogeneous group of disorders resulting in severe glomerularinflammation and injury. Clinically, RPGN is characterized by a rapidloss of glomerular filtration rate, haematuria, and proteinuria causedby characteristic glomerular lesions such as capillary necrosis andhyperplasia of the parietal epithelial cells (PEC) along Bowman'scapsule forming crescents.

The pathogenesis of RPGN involves autoantibodies, immunecomplex-mediated activation of complement, the local production ofcytokines and chemokines and glomerular leukocyte recruitment (Couser,2012, J Am Soc Nephrol 23:381). RPGN is more common in anti-neutrophilcytoplasmic antibody (ANCA)-associated GN or anti-glomerular basementmembrane (GBM) disease than in other forms of GN (Berden et al., 2010, JAm Soc Nephrol 21:1628; Jennette et al., 2006, J Am Soc Nephrol17:1235). The hallmark of severe GN is glomerular capillary necrosisleading to hematuria and plasma leakage (Bonsib, 1985, Am J Pathol119:357). PEC exposure to plasma is sufficient to trigger crescentformation (Ryu et al., 2012, J Pathol 228:382) but inflammation and PECinjury serve as additional stimuli (Sicking et al., 2012, J Am SocNephrol 23:629). The question of what causes vascular necrosis insidethe glomerulus has not previously been answered.

Severe glomerulonephritis involves cell necrosis as well as NETosis, aprogrammed neutrophil death leading to expulsion of nuclear chromatinleading to neutrophil extracellular traps (NETs). ETosis is a programmedform of cell death of mostly neutrophils (referred to as NETosis) andother granulocytes (Brinkmann et al., 2004, Science 303:1532). NETosiscauses an explosion-like directed expulsion of chromatin generating ameshwork called neutrophil extracellular traps (NETs), which immobilizeand kill bacteria during infections (Brinkmann et al., 2004, Science303:1532). Cytokine-induced NETosis also drives sterile injury includingnecrotizing GN (Kessenbrock et al., 2009, Nat Med 15:623; Kambas et al.,2013, Ann Rheum Dis 73:1854; Nakazawa et al., 2012, Front Immunol 3:333;Tsuboi et al., 2002, J Immunol 169:2026). Many cytosolic orchromatin-related components could account for the toxic andpro-inflammatory effect of NETs, such as proteolytic enzymes orintracellular molecules with immunostimulatory effects, referred to asdanger-associated molecular patterns (DAMPs) (Rock et al., 2010, AnnualReview of Immunology 28:321).

Histones are nuclear proteins that wind up the double-stranded DNA toform chromatin. Dynamic modifications of histone residues regulate genetranscription by determining the accessibility of transcription factorsto their DNA binding sites (Helin & Dhanak, 2013, Nature 502:480). Whencell necrosis releases histones into the extracellular space theydisplay significant cytotoxic effects (Hirsch, 1958, J Exp Med 108:925;Xu et al., 2009, Nat Med 15:1318; Chaput & Zychlinsky, 2009, Nat Med15:1245; Allam et al., 2014, J Mol Med 92:465). Histones contribute tofatal outcomes in murine endotoxinemia caused by microvascular injuryand activation of coagulation (Xu et al., 2009, Nat Med 15:1318; Abramset al., 2013, Am J Respir Crit Care Med 187:160; Saffarzadeh et al.,2012, PLoS One 7:e32366; Semeraro et al., 2011, Blood 118:1952). Wepreviously showed that dying renal cells release extracellular histonesthat promote septic and post-ischemic acute kidney injury (Allam et al.,2012, J Am Soc Nephrol 23:1375). We further demonstrated that histonesact as DAMPs by activating Toll-like receptor (TLR)-2 and -4 as well asNLRP3 (Allam et al., 2012, J Am Soc Nephrol 23:1375; Allam et al., 2013,Eur J Immunol 43:3336), which was confirmed by other groups (Semeraro etal., 2011, Blood 118:1952; Huang et al., 2013, J Immunol 191:2665; Xu etal., 2011, J Immunol 187:2626). TLR2/-4-mediated pathology is anessential mechanism of crescentic GN (Brown et al., 2006, J Immunol177:1925; Brown et al., 2007, J Am Soc Nephrol 18:1732).

A need exists for improved methods and compositions for treatment ofvascular necrosis in severe glomerulonephritis, preferably usinghistone-neutralizing agents such as anti-histone antibodies or fragmentsthereof.

SUMMARY

The present invention concerns compositions and methods of anti-histonetherapy for vascular necrosis in severe glomerulonephritis. Preferablythe anti-histone therapy may involve use of agents such as activatedprotein C, heparin, or anti-histone antibodies, such as antibodiesagainst histone H2B, H3 or H4. In more preferred embodiments, theanti-histone antibody may be a BWA-3 anti-H4 antibody (see, e.g., U.S.patent application Ser. No. 14/620,315, the Examples section and Figuresof which are incorporated herein by reference).

Preferably, the anti-histone antibodies or fragments thereof may bechimeric, humanized or human. The antibody can be of various isotypes,preferably human IgG1, IgG2, IgG3 or IgG4, more preferably comprisinghuman IgG1 hinge and constant region sequences. Most preferably, theantibody or fragment thereof may be designed or selected to comprisehuman constant region sequences that belong to specific allotypes, whichmay result in reduced immunogenicity when the immunoconjugate isadministered to a human subject. Preferred allotypes for administrationinclude a non-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 orG1m3,1,2. More preferably, the allotype is selected from the groupconsisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes. Exemplaryhumanized anti-histone antibodies are disclosed in U.S. patentapplication Ser. No. 14/620,315, the Figures and Examples section ofwhich are incorporated herein by reference.

In certain preferred embodiments, a combination of anti-histoneantibodies may be used. Antibodies against human histones H1, H2A, H2B,H3 or H4 may be used in any combination. Other non-antibody therapeuticagents targeted against either histones or downstream effectors of ahistone-mediated pathway may also be utilized in combination withanti-histone antibodies or fragments thereof, administered eitherbefore, simultaneously with, or following administration of one or moreanti-histone antibodies or fragments thereof. Various therapeutic agentsof use in treating histone-associated diseases are known in the art,such as activated protein C (APC), thrombomodulin, a peptide fragment ofhistone H1, H2A, H2B, H3 or H4, granzyme A, granzyme B, plasmin, Factor7-activating protease, heparin, and any such known agent may be utilizedin combination with anti-histone antibodies or antibody fragments. Ahuman histone H4 peptide may comprise residues 50-67 or 40-78 of humanH4 (see, e.g., U.S. Publ. No. 20090117099). Depending on the underlyingetiology, the anti-histone agents may also be utilized in combinationwith one or more standard treatments for glomerulonephritis and/orkidney failure, such as corticosteroids, immune-suppressing drugs orplasmapheresis.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate preferred embodimentsof the invention. However, the claimed subject matter is in no waylimited by the illustrative embodiments disclosed in the drawings.

FIG. 1A. TLR2 and TLR4 expression in human crescenticglomerulonephritis. Toll-like receptor (TLR)-2 and -4 immunostaining wasperformed on healthy kidney tissue. Original magnification ×400.

FIG. 1B. TLR2 and TLR4 expression in human crescenticglomerulonephritis. Toll-like receptor (TLR)-2 and -4 immunostaining wasperformed on kidney biopsies of a patient with newly diagnosed ANCAvasculitis and clinical signs of glomerulonephritis. FIG. 1B showsrepresentative glomeruli unaffected by loop necrosis or crescentformation. Original magnification ×400.

FIG. 1C. TLR2 and TLR4 expression in human crescenticglomerulonephritis. Toll-like receptor (TLR)-2 and -4 immunostaining wasperformed on kidney biopsies of a patient with newly diagnosed ANCAvasculitis and clinical signs of glomerulonephritis. FIG. 1C showsrepresentative glomeruli affected by loop necrosis or crescentformation. Original magnification ×400.

FIG. 1D. TLR2 and TLR4 expression in human crescenticglomerulonephritis. Toll-like receptor (TLR)-2 and -4 immunostaining wasperformed on kidney biopsies of a patient with newly diagnosed ANCAvasculitis and clinical signs of glomerulonephritis. FIG. 1D showsrepresentative glomeruli affected by loop necrosis or crescentformation. Original magnification ×400.

FIG. 2A. NETosis-related extracellular histones kill glomerular cells.Murine glomerular endothelial cells (GEnC), podocytes, and parietalepithelial cells (PECs) were incubated with increasing doses of histonestogether with either control IgG or anti-histone IgG. Cell viability wasdetermined after 24 hours by MTT assay. Data represent mean OD±SEM ofthree experiments measured at a wavelength of 570 nm. *p<0.05, **p<0.01,***p<0.001 versus control IgG.

FIG. 2B. NETosis-related extracellular histones kill glomerular cells.Immunostaining of naïve (left) and TNF-α-activated neutrophils (right)in culture. Staining for elastase (red) and histones (green) illustrateshow TNF-α triggers NETosis leading to neutrophil extracellular trap(NET) formation, i.e. expelling cytoplasmic and nuclear contents in theextracellular space.

FIG. 2C. NETosis-related extracellular histones kill glomerular cells.Scanning electron microscopy was performed on monolayers of glomerularendothelial cells, which appear flat and evenly laid out on scanningelectron microscopy (left). However, neutrophil ETosis leads to severeinjury and death of endothelial cells appearing as bulging white ballswith corrugated surfaces adjacent to activated NETs (middle). Thiseffect was almost entirely prevented by anti-histone IgG demonstrated bysignificant reversal of the structural integrity of the endothelial cellmonolayer (right).

FIG. 2D. NETosis-related extracellular histones kill glomerular cells.Immunostaining for elastase, histone, and DAPI was performed onmonolayers of glomerular endothelial cells. Conditions were as stated atthe bottom of the Figure.

FIG. 2E. NETosis-related extracellular histones kill glomerular cells.MTT assay analysis of endothelial cell viability allowed to quantifythis effect, which was identical for TNF-α and PMA, two known inducersof NETosis. Data represent mean OD±SEM of three experiments measured ata wavelength of 570 nm. *p<0.05, **p<0.01, ***p<0.001 versus controlIgG.

FIG. 3A. Extracellular histones injure glomeruli in a TLR2/4-dependentmanner. Glomeruli were isolated from wild type and Tlr2/4-deficient miceand incubated with histones (30 μg/ml). After 12 hours LDH release intothe supernatant was measured as a marker of glomerular cell injury. Datarepresent mean OD±SEM of three experiments measured at a wavelength of492 nm. **p<0.01, ***p<0.001 versus control.

FIG. 3B. Extracellular histones injure glomeruli in a TLR2/4-dependentmanner. For intra-arterial histone injection the abdominal aorta wasprepared and a micro-cannula was placed into the left renal artery toinject histones directly into the kidney. Images show hematoxylin-eosinstaining of representative glomeruli of the different groups asindicated.

FIG. 3C. Extracellular histones injure glomeruli in a TLR2/4-dependentmanner. Fibrinogen immunostaining displayed three different stainingpatterns. FIG. 3C shows diffuse positivity of glomerular endothelialcells.

FIG. 3D. Extracellular histones injure glomeruli in a TLR2/4-dependentmanner. Fibrinogen immunostaining displayed three different stainingpatterns. FIG. 3D shows entire luminal positivity indicatingmicrothrombus formation.

FIG. 3E. Extracellular histones injure glomeruli in a TLR2/4-dependentmanner. Fibrinogen immunostaining displayed three different stainingpatterns. FIG. 3E shows global positivity of glomerular loop indicatingloop necrosis.

FIG. 3F. Extracellular histones injure glomeruli in a TLR2/4-dependentmanner. A quantitative analysis of these lesions revealed that histoneinjection massively increased luminal and global fibrinogen positivity,which was partially prevented in Tlr2/4-deficient mice. **p<0.01,***p<0.001 versus saline, #p<0.05, ##p<0.01 versus histone group.

FIG. 4A. Neutralizing histones protects from severe glomerulonephritis.Blood urea nitrogen (BUN) levels were determined 1 and 7 days afterintravenous injection of GBM antiserum. Mice were either treated withcontrol IgG or anti-histone IgG starting from the day before antiseruminjection.

FIG. 4B. Neutralizing histones protects from severe glomerulonephritis.Representative HE stainings of glomeruli are shown at an originalmagnification of 400×.

FIG. 4C. Neutralizing histones protects from severe glomerulonephritis.Morphometrical analysis of segmental and global glomerular lesions(left) and of glomeruli with crescents (right) as described in methods.

FIG. 4D. Neutralizing histones protects from severe glomerulonephritis.CD31 and MPO immumostaining representing NETs formation in the glomeruliclose association with the endothelial cells, control IgG group showsfocal loss of endothelial cell positivity compare to anti-histone IgGgroup. Data means±SEM from five to six mice in each group. *p<0.05,**p<0.01, ***p<0.001 versus control IgG.

FIG. 5A. Neutralizing histones protects the glomerular filtrationbarrier in glomerulonephritis. Transmission electron microscopy ofantiserum-induced GN revealed extensive glomerular injury with fibrinoidnecrosis (upper left), endothelial cell swelling, luminal thrombosis,and intraluminal granulocytes (upper middle and right). Podocytes showfoot process effacement (all upper images). Pre-emptive treatment withanti-histone IgG decreased most of these abnormalities; particularlyendothelial cell and podocyte ultrasturcture (lower images).

FIG. 5B. Neutralizing histones protects the glomerular filtrationbarrier in glomerulonephritis. Immunostaining for WT-1 (red) and nephrin(green) was used to quantify podocytes.

FIG. 5C. Neutralizing histones protects the glomerular filtrationbarrier in glomerulonephritis. Anti-histone IgG doubled the number ofnephrin/WT-1+ podocytes at day 7 of antiserum-induced GN.

FIG. 5D. Neutralizing histones protects the glomerular filtrationbarrier in glomerulonephritis. Urinary albumin/creatinine ratio wasdetermined at day 1 and day 7 after antiserum injection. Data representmean±SEM from five to six mice of each group. *p<0.05, ***p<0.001 versuscontrol IgG.

FIG. 6A. Leukocyte recruitment and activation in glomerulonephritis.Glomerular neutrophil and macrophage infiltrates were quantified byimmunostaining. Representative images are shown at an originalmagnification of 400×.

FIG. 6B. Leukocyte recruitment and activation in glomerulonephritis.Leukocyte activation was quantified by flow cytometry of renal cellsuspensions harvested 7 days after antiserum injection. Data representmean±SEM from five to six mice of each group.

FIG. 6C. Leukocyte recruitment and activation in glomerulonephritis.Cultured dendritic cells were exposed to increasing doses of histones asindicated. After 24 h flow cytometry was used to determine thepercentage of cells that express the activation markers MHC II, CD40,CD80, and CD86. Data are means±SEM from three independent experiments.*p<0.05, **p<0.01, ***p<0.001 versus control IgG.

FIG. 7A. Histones activate TNF-α production. Cultured J774 macrophagesand bone marrow dendritic cells (BMDCs) respond to histone exposure byinducing the secretion of TNF-α, which is blocked by anti-histone IgG.Data are means±SEM from three independent experiments. ***p<0.001 versuscontrol IgG.

FIG. 7B. Histones activate TNF-α production. TNF-α immunostaining onrenal sections from both treatment groups taken at day 7 after antiseruminjection. Representative images are shown at an original magnificationof 400×.

FIG. 7C. Histones activate TNF-α production. Real time RT-PCR for TNF-αmRNA on renal tissue at day 7 after antiserum injection. Data aremeans±SEM from at least five to six mice in each group. *p<0.05 versuscontrol IgG.

FIG. 7D. Histones activate TNF-α production. Fibrinogen immunostainingon renal sections from both treatment groups taken at day 7 afterantiserum injection. Representative images are shown at an originalmagnification of 400×.

FIG. 7E. Histones activate TNF-α production. Real time RT-PCR forfibrinogen mRNA on renal tissue at day 7 after antiserum injection. Dataare means±SEM from at least five to six mice in each group. *p<0.05versus control IgG.

FIG. 7F. Histones activate TNF-a production. Immunostaining of renalsections of all groups for claudin-1 (red, marker for parietalepithelial cells/PECs and some tubular cells), WT-1 (green, marker forpodocytes and activated PECs), and DAPI (blue, DNA marker) illustratesthat in severe GN crescents consist of WT-1+ PECs, which is reversedwith anti-histone IgG. Original magnification: ×200.

FIG. 7G. Histones activate TNF-α production. Mouse PEC viability (MTTassay) when cultured in the presence of different serum concentrationstogether with a low concentration (20 μg/ml) of histones that withoutserum reduces PEC viability. Together with serum histones rather promotePEC growth.

FIG. 7H. Histones activate TNF-α production. Mouse PEC viability (MTTassay) experiment showing that blocking anti-TLR2 and anti-TLR4antibodies neutralize the histone effect on PEC growth. Data are meanOD±SEM of three experiments measured at a wavelength of 570 nm.**p<0.01, ***p<0.001 versus control IgG.

FIG. 7I. Histones activate TNF-α production. RT-PCR analysis of PECsstimulated with histones and various neutralizing compounds(anti-histone IgG, heparin 50 μg/ml, activated protein C 500 nM,anti-TLR2 or -4 1 ng/ml). Note that all these interventions blockhistone-induced CD44 and WT-1 mRNA expression, which serve as markers ofPEC activation. Data are means±SEM of three experiments. *p<0.05,**p<0.01, ***p<0.001 versus histone group.

FIG. 8A. Delayed histone blockade still improves glomerulonephritis.Glomeruli were isolated from wild type mice and incubated with histonesin the presence or absence of anti-histone IgG, heparin or aPC asbefore. LDH release was measured in supernatants as a marker ofglomerular cell injury. Data are mean OD±SEM of three experiments.*p<0.05, **p<0.01, ***p<0.001 versus control IgG or vehicle grouphistone group, respectively.

FIG. 8B. Delayed histone blockade still improves glomerulonephritis.Further experiments used the model of antiserum-induced GN usinganti-histone IgG, heparin or recombinant aPC initiated only afterdisease onset, i.e. 24 h after antiserum injection, when the urinaryalbumin/creatinine ratio was around 80 μg/mg.

FIG. 8C. Delayed histone blockade still improves glomerulonephritis.Data show plasma creatinine levels at day 7 and albuminuria also at day2.

FIG. 8D. Delayed histone blockade still improves glomerulonephritis.Podocytes were quantified as nephrin/WT-1+ cells on renal sections atday 7.

FIG. 8E. Delayed histone blockade still improves glomerulonephritis.Glomerular lesions were quantified by morphometry from PAS sectionstaken at day 7. Glomerular podocyte numbers were assessed byWT-1/nephrin co-staining on glomerular cross sections. Data representmean±SEM from five to six mice of each group. *p<0.05, **p<0.01,***p<0.001 versus control IgG or vehicle, respectively.

FIG. 8F. Delayed histone blockade still improves glomerulonephritis.Glomerular crescents were quantified by morphometry from PAS sectionstaken at day 7. Glomerular podocyte numbers were assessed byWT-1/nephrin co-staining on glomerular cross sections. Data representmean±SEM from five to six mice of each group. *p<0.05, **p<0.01,***p<0.001 versus control IgG or vehicle, respectively.

FIG. 8G. Delayed histone blockade still improves glomerulonephritis.Tubular injury was quantified by morphometry from PAS sections taken atday 7. Glomerular podocyte numbers were assessed by WT-1/nephrinco-staining on glomerular cross sections. Data represent mean±SEM fromfive to six mice of each group. *p<0.05, **p<0.01, ***p<0.001 versuscontrol IgG or vehicle, respectively.

FIG. 9A. Histones and endothelial cell microtubes in vitro. Murineglomerular endothelial cells were seeded into a matrigel matrix forangiogenesis experiments as described in methods. Histones +/−anti-histone IgG were added during microtube formation to test histonetoxicity on microtube forming. Quantitative assessment included thenumber of living cells and network formation. Data represent the mean ofthese endpoints±SEM of three independent experiments at the indicatedtime points. *p<0.05, **p<0.01, ***p<0.001 versus control IgG.

FIG. 9B. Histones and endothelial cell microtubes in vitro. Murineglomerular endothelial cells were seeded into a matrigel matrix forangiogenesis experiments as described in methods. Histones +/−anti-histone IgG were added 8 h after microtube formation to testhistone toxicity on microtube destruction. Quantitative assessmentincluded the number of living cells and network formation. Datarepresent the mean of these endpoints±SEM of three independentexperiments at the indicated time points. *p<0.05, **p<0.01, ***p<0.001versus control IgG.

FIG. 10. Anti-histone IgG and podocyte detachment in vitro. Murinepodocytes were exposed to histones or GBM antiserum with or withoutanti-histone IgG. Data show the mean percentage±SEM of podocytes thatdetached from the culture dish within 24 hours. *p<0.05, ***p<0.001versus control.

FIG. 11A. Cytokine induction in glomeruli ex vivo exposed to histones.Glomeruli were isolated from wild type mice and Tlr2-/4 double knockoutmice and exposed to histones. 12 hours later the mRNA levels of TNF-αwere determined by real-time RT-PCR. Data show the mRNA expression levelcorrected for the housekeeper 18srRNA±SEM each done in triplicate.*p<0.05, **p<0.01, n.s.=not significant.

FIG. 11B. Cytokine induction in glomeruli ex vivo exposed to histones.Glomeruli were isolated from wild type mice and Tlr2-/4 double knockoutmice and exposed to histones. 12 hours later the mRNA levels of IL-6were determined by real-time RT-PCR. Data show the mRNA expression levelcorrected for the housekeeper 18srRNA±SEM each done in triplicate.*p<0.05, **p<0.01, n.s.=not significant.

FIG. 11C. Cytokine induction in glomeruli ex vivo exposed to histones.Glomeruli were isolated from wild type mice and Tlr2-/4 double knockoutmice and exposed to histones. IL-6 protein release was determined incell culture supernatants. *p<0.05, **p<0.01, n.s.=not significant.

FIG. 12A. MIP2/CXCXL2 mRNA expression in glomerular endothelial cells(GEnC). Seven days after sheep GBM antiserum injection in vivo onlysheep IgG (left) deposits were found in glomeruli but no mouse IgG.

FIG. 12B. MIP2/CXCXL2 mRNA expression in glomerular endothelial cells(GEnC). Exposure to glomerular basement membrane (GBM) antiserum inducesthe mRNA expression levels of MIP2/CXCL2. Data are means±SEM from threeindependent experiments.

FIG. 13A. Heparin and activated protein C (aPC) block histone toxicityon glomerular endothelial cells. Glomerular endothelial cells wereexposed to increasing doses of histones with or without heparin asindicated. Data represent mean OD±SEM of three MTT assay experimentsmeasured at a wave length of 492 nm.

FIG. 13B. Heparin and activated protein C (aPC) block histone toxicityon glomerular endothelial cells. Glomerular endothelial cells wereexposed to increasing doses of histones with or without aPC asindicated. Data represent mean OD±SEM of three MTT assay experimentsmeasured at a wave length of 492 nm.

FIG. 14A. Therapeutic histone blockade and renal leukocytes. Renalsections were obtained at day 7 of the experiment and stained for GR-1(neutrophils). Data represent mean glomerular cell counts±SEM of 5-6mice in each group.

FIG. 14B. Therapeutic histone blockade and renal leukocytes. Renalsections were obtained at day 7 of the experiment and stained for Mac2(macrophages). Data represent mean glomerular cell counts±SEM of 5-6mice in each group.

FIG. 14C. Therapeutic histone blockade and renal leukocytes. Renalsections were obtained at day 7 of the experiment. Flow cytometry datafor various leukocyte subsets as indicated. *p<0.05, **p<0.01,***p<0.001 versus control IgG or vehicle, respectively.

FIG. 14D. Therapeutic histone blockade and renal leukocytes. Renalsections were obtained at day 7 of the experiment. Flow cytometry datafor various leukocyte subsets as indicated. *p<0.05, **p<0.01,***p<0.001 versus control IgG or vehicle, respectively.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, “a” or “an” means “one or more”.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, peptides, drugs, toxins, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MRI).

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody). An “antibody” includes monoclonal, polyclonal, bispecific,multispecific, murine, chimeric, humanized and human antibodies.

A “naked antibody” is an antibody or antigen binding fragment thereofthat is not attached to a therapeutic or diagnostic agent. The Fcportion of an intact naked antibody can provide effector functions, suchas complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev50:59-87, 1998). Other mechanisms by which naked antibodies induce celldeath may include apoptosis. (Vaswani and Hamilton, Ann Allergy AsthmaImmunol 81: 105-119, 1998.)

An “antibody fragment” is a portion of an intact antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv, scFv, dAb and the like. Regardlessof structure, an antibody fragment binds with the same antigen that isrecognized by the full-length antibody. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains or recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). “Single-chain antibodies”, often abbreviated as“scFv” consist of a polypeptide chain that comprises both a V_(H) and aV_(L) domain which interact to form an antigen-binding site. The V_(H)and V_(L) domains are usually linked by a peptide of 1 to 25 amino acidresidues. Antibody fragments also include diabodies, triabodies andsingle domain antibodies (dAb). Fragments of antibodies that do not bindto the same antigen as the intact antibody, such as the Fc fragment, arenot included within the scope of an “antibody fragment” as used herein.

A “chimeric antibody” is a recombinant protein that contains thevariable domains of both the heavy and light antibody chains, includingthe complementarity determining regions (CDRs) of an antibody derivedfrom one species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A “humanized antibody” is a recombinant protein in which the CDRs froman antibody from one species; e.g., a murine antibody, are transferredfrom the heavy and light variable chains of the murine antibody intohuman heavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A “human antibody” is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

An “immunoconjugate” is an antibody, antigen-binding antibody fragment,antibody complex or antibody fusion protein that is conjugated to atherapeutic agent. Conjugation may be covalent or non-covalent.Preferably, conjugation is covalent.

As used herein, the term “antibody fusion protein” is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An “immunomodulator” is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

An anti-histone antibody or antibody fragment, or a compositiondescribed herein, is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient subject.In particular embodiments, an antibody preparation is physiologicallysignificant if its presence invokes an antitumor response or mitigatesthe signs and symptoms of an autoimmune disease state. A physiologicallysignificant effect could also be the evocation of a humoral and/orcellular immune response in the recipient subject leading to growthinhibition or death of target cells.

Anti-Histone Antibodies

Various anti-histone antibodies and/or antigen-binding fragments thereofmay be of use. The murine BWA-3 (anti-H4), LG2-1 (anti-H3) and LG2-2(anti-H2B) hybridomas were reported by Monestier et al. (1993, Mol.Immunol 30:1069-75). However, murine antibodies are generally notappropriate for human therapeutic use, due to the formation of humananti-mouse antibodies (HAMA) that can neutralize these anatibodies andthus make them less active.

In preferred embodiments, a humanized or chimeric anti-histone H4antibody is one that comprises the heavy chaincomplementarity-determining region (CDR) sequences CDR1 (DDYLH, SEQ IDNO:90), CDR2 (WIGWIDPENGDTEYASKFQG, SEQ ID NO:91) and CDR3 (PLVHLRTFAY,SEQ ID NO:92) and the light chain CDR sequences CDR1 (RASESVDSYDNSLH,SEQ ID NO:93), CDR2 (LASNLES, SEQ ID NO:94) and CDR3 (QQNNEDPWT, SEQ IDNO:95). (See, e.g., U.S. Pat. No. 8,987,421, the Figures and Examplessection of which are incorporated herein by reference.)

In other preferred embodiments, a humanized or chimeric anti-histoe H3antibody is one that comprises the heavy chain CDR sequences CDR1(SYWMH, SEQ ID NO:96), CDR2 (NIDPSDSETHYNQKFKD, SEQ ID NO:97) and CDR3(EKITDDYNYFDY, SEQ ID NO:98) and the light chain CDR sequences CDR1(RASESVDSYGNSFMH, SEQ ID NO:99), CDR2 (HASNLES, SEQ ID NO:100) and CDR3(QQNNEDPLT, SEQ ID NO:101) (see, e.g., U.S. Pat. No. 8,987,421).

In still other preferred embodiments, a humanized or chimericanti-histone H2B antibody is one that comprises the heavy chain CDRsequences CDR1 (SYVMY, SEQ ID NO:102), CDR2 (YINPYNDGTKYNEKFKG, SEQ IDNO:103) and CDR3 (PGDGYPFDY, SEQ ID NO:104) and the light chain CDRsequences CDR1 (RSSQSIVHSNGNTYLE, SEQ ID NO:105), CDR2 (KVSNRFS, SEQ IDNO:106) and CDR3 (FQGSHVPYT, SEQ ID NO:107) (see, e.g., U.S. Pat. No.8,987,421).

General Techniques for Antibodies and Antibody Fragments

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). The person of ordinary skill may readily produce antibodiesagainst any known and characterized target antigen, using only routineexperimentation. Known antigens that may be targeted include, but arenot limited to, human histone H4 (e.g., NCBI Ref. No. NP_778224.1),human histone H3 (e.g., GenBank Ref. No. CAB02546.1) or human histoneH2B (e.g., GenBank Ref. No. CAB02542.1)

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal.,

“Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XenoMouse® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

Techniques for producing single domain antibodies are also known in theart, as disclosed for example in Cossins et al. (2006, Prot ExpressPurif 51:253-259), incorporated herein by reference. Single domainantibodies (VHH) may be obtained, for example, from camels, alpacas orIlamas by standard immunization techniques. (See, e.g., Muyldermans etal., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75,2003; Maass et al., J Immunol Methods 324:13-25, 2007). The VHH may havepotent antigen-binding capacity and can interact with novel epitopesthat are inacessible to conventional VH-VL pairs. (Muyldermans et al.,2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgGantibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized withknown antigens, such as TNF-α, and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). In certainembodiments, anti-pancreatic cancer VHH antibody fragments may beutilized in the claimed compositions and methods.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and/orhave published variable region sequences and are available for use inthe claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572;856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206′ 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Particular antibodies that may be of use include, but are not limitedto, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e), hL243(anti-HLA-DR), alemtuzumab (anti-CD52), gemtuzumab (anti-CD33),ibritumomab tiuxetan (anti-CD20); rituximab (anti-CD20); tositumomab(anti-CD20); and GA101 (anti-CD20). Such antibodies are known in the art(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. PatentApplication Publ. No. 20040202666 (now abandoned); 20050271671; and20060193865; the Examples section of each incorporated herein byreference.) Specific known antibodies of use include hA20 (U.S. Pat. No.7,251,164), hA19 (U.S. Pat. No. 7,109,304), hLL1 (U.S. Pat. No.7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hL243 (U.S. Pat. No.7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15 (U.S. Pat. No.7,541,440), and hMN-3 (U.S. Pat. No. 7,541,440), the text of eachrecited patent or application is incorporated herein by reference withrespect to the Figures and Examples sections.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as autoimmunediseases like rheumatoid arthritis and systemic lupus erythematosus(Morand & Leech, 2005, Front Biosci 10:12-22; Shachar & Haran, 2011,Leuk Lymphoma 52:1446-54); kidney diseases such as renal allograftrejection (Lan, 2008, Nephron Exp Nephrol. 109:e79-83); and numerousinflammatory diseases (Meyer-Siegler et al., 2009, Mediators Inflammepub Mar. 22, 2009; Takahashi et al., 2009, Respir Res 10:33;Milatuzumab (hLL1) is an exemplary anti-CD74 antibody of therapeutic usefor treatment of MIF-mediated diseases.

Bispecific and Multispecific Antibodies

Bispecific or multispecific antibodies can be prepared by a variety ofprocedures, ranging from glutaraldehyde linkage to more specificlinkages between functional groups. The antibodies and/or antibodyfragments are preferably covalently bound to one another, directly orthrough a linker moiety, through one or more functional groups on theantibody or fragment, e. g., amine, carboxyl, phenyl, thiol, or hydroxylgroups. Various conventional linkers in addition to glutaraldehyde canbe used, e. g., disiocyanates, diiosothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides, maleimidehydroxy-succinimdeesters, and the like. The optimal length of the linker may varyaccording to the type of target cell.

A simple method to produce multivalent antibodies is to mix theantibodies or fragments in the presence of glutaraldehyde. The initialSchiff base linkages can be stabilized, e. g., by borohydride reductionto secondary amines. A diiosothiocyanate or carbodiimide can be used inplace of glutaraldehyde as a non-site-specific linker.

The simplest form of a multivalent, multispecific antibody is abispecific antibody. Bispecific antibodies can be made by a variety ofconventional methods, e. g., disulfide cleavage and reformation ofmixtures of whole IgG or, preferably F (ab′)₂ fragments, fusions of morethan one hybridoma to form polyomas that produce antibodies having morethan one specificity, and by genetic engineering. Bispecific antibodieshave been prepared by oxidative cleavage of Fab′ fragments resultingfrom reductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F (ab′)₂ fragments produced bypepsin digestion of two different antibodies, reductive cleavage to forma mixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F (ab′)₂ fragments includingbispecific antibodies containing a Fab′ portion specific to each of theoriginal epitopes.

General techniques for the preparation of multivalent antibodies may befound, for example, in Nisonphoff et al., Arch Biochem. Biophys. 93: 470(1961), Hammerling et al., J. Exp. Med. 128: 1461 (1968), and U.S. Pat.No. 4,331,647.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimide-hydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g., an antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e. g., Traut's Reagent. Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen binding sites. This can be accomplished by, e. g.,linkage to cleaved interchain sulfydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody which has at least one free aminefunction. This results in an initial Schiff base (imine) linkage, whichis preferably stabilized by reduction to a secondary amine, e. g., byborohydride reduction, to form the final product. Such site-specificlinkages are disclosed, for small molecules, in U.S. Pat. No. 4,671,958,and for larger addends in U.S. Pat. No. 4,699,784.

Alternatively, such bispecific antibodies can be produced by fusing twohybridoma cell lines that produce appropriate Mabs. Techniques forproducing tetradomas are described, for example, by Milstein et al.,Nature 305: 537 (1983) and Pohl. et al., Int. J. Cancer 54: 418 (1993).

Alternatively, chimeric genes can be designed that encode both bindingdomains. General techniques for producing bispecific antibodies bygenetic engineering are described, for example, by Songsivilai et al.,Biochem Biophys Res. Commun 164: 271 (1989); Traunecker et al., EMBO J.10: 3655 (1991); and Weiner et al., J. Immunol. 147: 4035 (1991).

A higher order multivalent, multispecific molecule can be obtained byadding various antibody components to a bispecific antibody, produced asabove. For example, a bispecific antibody can be reacted with2-iminothiolane to introduce one or more sulfhydryl groups for use incoupling the bispecific antibody to a further antibody derivative thatbinds an the same or a different epitope of the target antigen, usingthe bis-maleimide activation procedure described above. These techniquesfor producing multivalent antibodies are well known to those of skill inthe art. See, for example, U.S. Pat. No. 4,925,648, and Goldenberg,international publication No. WO 92/19273, which are incorporated byreference.

DOCK-AND-LOCK™ (DNL™)

In preferred embodiments, a bispecific or multispecific antibody isformed as a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos.7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examplessection of each of which is incorporated herein by reference.)Generally, the technique takes advantage of the specific andhigh-affinity binding interactions that occur between a dimerization anddocking domain (DDD) sequence of the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequencederived from any of a variety of AKAP proteins (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). The DDD and AD peptides may be attached to any protein,peptide or other molecule. Because the DDD sequences spontaneouslydimerize and bind to the AD sequence, the technique allows the formationof complexes between any selected molecules that may be attached to DDDor AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968;243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991;50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990;265;21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004;5:959). The AD of AKAPs for PKA isan amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991;266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003;100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999;6:222; Newlon et al., EMBO J.2001;20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480)to ligate site-specifically. Using various combinations of linkers,adaptor modules and precursors, a wide variety of DNL™ constructs ofdifferent stoichiometry may be produced and used (see, e.g., U.S. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL™construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 1. In devising Table 1, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 1 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 87. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V VTHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 12)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 2 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 1 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 2 Conservative Amino Acid Substitutions in AD1(SEQ ID NO: 3). Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y LA K Q I V D N A I Q Q A N L D F I R N E Q N N L V T V I S VNIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO: 49)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA(SEQ ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

II-Specific AKAPs AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 55) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 57)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 59) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 63) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 3 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 3 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31 KGADLIEEAASRIVDAVIEQVKAAG(SEQ ID NO: 68) Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69)AKAP7δ-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A 

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 4. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 1 andTable 2.

TABLE 4 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO:1).Consensus sequence disclosed as SEQ ID NO: 89. S H I Q I P P G L T E L LQ G Y T V E V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A RA N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Alternative DNL™ Structures

In certain alternative embodiments, DNL™ constructs may be formed usingalternatively constructed antibodies or antibody fragments, in which anAD moiety may be attached at the C-terminal end of the kappa light chain(C_(k)), instead of the C-terminal end of the Fc on the heavy chain. Thealternatively formed DNL™ constructs may be prepared as disclosed inProvisional U.S. patent application Ser. Nos. 61/654,310, filed Jun. 1,2012, 61/662,086, filed Jun. 20, 2012, 61/673,553, filed Jul. 19, 2012,and 61/682,531, filed Aug. 13, 2012, the entire text of eachincorporated herein by reference. The light chain conjugated DNL™constructs exhibit enhanced Fc-effector function activity in vitro andimproved pharmacokinetics, stability and anti-lymphoma activity in vivo(Rossi et al., 2013, Bioconjug Chem 24:63-71).

C_(k)-conjugated DNL™ constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. Nos. 61/654,310, 61/662,086,61/673,553, and 61/682,531. Briefly, C_(k)-AD2-IgG, was generated byrecombinant engineering, whereby the AD2 peptide was fused to theC-terminal end of the kappa light chain. Because the natural C-terminusof C_(K) is a cysteine residue, which forms a disulfide bridge toC_(H)1, a 16-amino acid residue “hinge” linker was used to space the AD2from the C_(K)-V_(H)1 disulfide bridge. The mammalian expression vectorsfor C_(k)-AD2-IgG-veltuzumab and C_(k)-AD2-IgG-epratuzumab wereconstructed using the pdHL2 vector, which was used previously forexpression of the homologous C_(H)3-AD2-IgG modules. A 2208-bpnucleotide sequence was synthesized comprising the pdHL2 vector sequenceranging from the Bam HI restriction site within the V_(K)/C_(K) intronto the Xho I restriction site 3′ of the C_(k) intron, with the insertionof the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ IDNO:108) and AD2, in frame at the 3′end of the coding sequence for C_(K).This synthetic sequence was inserted into the IgG-pdHL2 expressionvectors for veltuzumab and epratuzumab via Bam HI and Xho I restrictionsites. Generation of production clones with SpESFX-10 were performed asdescribed for the C_(H)3-AD2-IgG modules. C_(k)-AD2-IgG-veltuzumab andC_(k)-AD2-IgG-epratuzumab were produced by stably-transfected productionclones in batch roller bottle culture, and purified from the supernatantfluid in a single step using MabSelect (GE Healthcare) Protein Aaffinity chromatography.

Following the same DNL process described previously for 22-(20)-(20)(Rossi et al., 2009, Blood 113:6161-71), C_(k)-AD2-IgG-epratuzumab wasconjugated with C_(H)1-DDD2-Fab-veltuzumab, a Fab-based module derivedfrom veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the 22*indicates the C_(k)-AD2 module of epratuzumab and each (20) symbolizes astabilized dimer of veltuzumab Fab. The properties of 22*-(20)-(20) werecompared with those of 22-(20)-(20), the homologous Fc-bsHexAbcomprising C_(H)3-AD2-IgG-epratuzumab, which has similar composition andmolecular size, but a different architecture.

Following the same DNL process described previously for 20-2b (Rossi etal., 2009, Blood 114:3864-71), C_(k)-AD2-IgG-veltuzumab, was conjugatedwith IFNα2b-DDD2, a module of IFNα2b with a DDD2 peptide fused at itsC-terminal end, to generate 20*-2b, which comprises veltuzumab with adimeric IFNα2b fused to each light chain. The properties of 20*-2b werecompared with those of 20-2b, which is the homologous Fc-IgG-IFNα.

Each of the bsHexAbs and IgG-IFNα were isolated from the DNL reactionmixture by MabSelect affinity chromatography. The two C_(k)-derivedprototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprisingepratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and aCD20-targeting immunocytokine, comprising veltuzumab and four moleculesof interferon-α2b, displayed enhanced Fc-effector functions in vitro, aswell as improved pharmacokinetics, stability and anti-lymphoma activityin vivo, compared to their Fc-derived counterparts.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(-1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain y-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1 m1 and nG1m1 allotype antibodies is shown forthe exemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab (SEQ IDNO:86).

Rituximab heavy chain variable region sequence  (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 86) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotypoe characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3  R 3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Exemplary antibody constant region sequences of use in the chimeric andhumanized anti-histone antibodies are disclosed in SEQ ID NO:109 and SEQID NO:110 below.

Exemplary human heavy chain constant region (SEQ ID NO: 109)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK  Exemplary human light chain constant region(SEQ ID NO: 110) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

Immunoconjugates

In certain embodiments, the antibodies or fragments thereof may beconjugated to one or more therapeutic or diagnostic agents. Thetherapeutic agents do not need to be the same but can be different, e.g.a drug and a radioisotope. For example, ¹³¹I can be incorporated into atyrosine of an antibody or fusion protein and a drug attached to anepsilon amino group of a lysine residue. Therapeutic and diagnosticagents also can be attached, for example to reduced SH groups and/or tocarbohydrate side chains. Many methods for making covalent ornon-covalent conjugates of therapeutic or diagnostic agents withantibodies or fusion proteins are known in the art and any such knownmethod may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.Pat. No. 7,563,433, the Examples section of which is incorporated hereinby reference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MRI. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F-Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

Therapeutic Agents

In alternative embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes orother agents may be used, either conjugated to the subject anti-histoneantibodies or other histone-neutralizing agents, or else separatelyadministered before, simultaneously with, or after thehistone-neutralizing agent. Drugs of use may possess a pharmaceuticalproperty selected from the group consisting of antimitotic, antikinase(e.g., anti-tyrosine kinase), alkylating, antimetabolite, antibiotic,alkaloid, anti-angiogenic, pro-apoptotic agents, immune modulators, andcombinations thereof.

Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine,anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. Lenolidamide is yet another immunomodulator that has shownactivity in controlling certain cancers, such as multiple myeloma andhematopoietic tumors.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2. A preferred formof therapeutic oligonucleotide is siRNA.

Immune Dysregulatory Disease

In various embodiments, the histone-neutralizing agents are of use totreat immune-dysregulatory diseases, such as glomerulonephritis. Incertain preferred embodiments, the therapy may utilize either acombination of two or more histone-neutralizing agents.

Additional therapeutic agents that may be added in combination include acytokine, a chemokine, a coagulation inhibitor, an anti-T cell or antiB-cell antibody or antibody fragment, an immunomodulator, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor, an interferon, erythropoietin or thrombopoietin. Anoptional therapeutic agent may include activated protein C, heparin orthrombomodulin, as mentioned above. Combinations of anti-histoneantibodies or fragments thereof with other histone neutralizing agents,including but not limited to antibodies or antibody fragments againstadditional immune system target antigens, as discussed below, may beutilized in certain embodiments.

The immune system comprises both the innate immune system and theadaptive or acquired immune system. Many host cells participate in theprocesses of innate and adaptive immunity, such as neutrophils, T- andB-lymphocytes, macrophages and monocytes, dendritic cells, and plasmacells. They usually act in concert, affecting one another, particularlyin the regulation of certain factors and cytokines that contribute tothe recognition and processing of innate and external noxients, andthese systems have evolved over the millions of years of the developmentof vertebrate, mammalian, and human organisms.

A major goal of immunotherapy is to exploit or enhance a patient'simmune system against an innate or foreign noxient, such as a malignantcell or an invading microorganism. The immune system has been studiedmore in relation to recognizing and responding to exogenous noxients,such as microbial organisms, than it has in relation to indigenousmalfunctions, such as cancer and certain autoimmune andimmune-dysregulatory diseases, particularly since the latter may haveboth genetic as well as environmental components. The defenses againstmicrobial organisms, such as bacteria, fungi, parasites, and viruses,are innate to the particular organism, with the immune system beingprogrammed to recognize biochemical patterns of these microorganisms andto respond to attack them without requiring prior exposure to themicroorganism. This innate immune system includes, for example,neutrophils, natural killer cells and monocytes/macrophages that caneradicate the invading microorganisms by direct engulfment anddestruction.

The innate immune response is often referred to as a nonspecific onethat controls an invading external noxient until the more specificadaptive immune system can marshal specific antibodies and T cells (cf.Modlin et al., N Engl J Med 1999, 340:1834-1835; Das, Crit. Care 2000;4:290-296). The nonspecific immune responses involve the lymphaticsystem and phagocytes. The lymphatic system includes the lymphocytes andmacrophages. Macrophages can engulf, kill and dispose of foreignparticles. Phagocytes include neutrophils and macrophages, which againingest, degrade and dispose of debris, and have receptors for complementand antibody. In summary, the innate immune system provides a line ofdefense again certain antigens because of inherited characteristics.

In contrast, the adaptive, or acquired, immune system, is highly evolvedand very specific in its responses. It is called an adaptive systembecause it occurs during the lifetime of an individual as an adaptationto infection with a pathogen. Adaptive immunity can be artificiallyacquired in response to a vaccine (antigens) or by administeringantibodies, or can be naturally acquired by infection. The acquiredimmunity can be active, if an antibody was produced, or it can bepassive, if exogenous antibody made from another source is injected.

The adaptive immune system produces antibodies specific to a givenantigen. The simplest and most direct way in which antibodies provideprotection is by binding to them and thereby blocking their access tocells that they may infect or destroy. This is known as neutralization.Binding by antibodies, however, is not sufficient to arrest thereplication of bacteria that multiply outside cells. In this case, onerole of antibody is to enable a phagocytic cell to ingest and destroythe bacterium. This is known as opsonization. The third function ofantibodies is to activate a system of plasma proteins, known ascomplement. In many cases, the adaptive immune system confers lifelongprotective immunity to re-infection with the same pathogen, because theadaptive immune system has a ‘memory’ of the antigens presented to it.

Antibody-mediated immunity is called humoral immunity and is regulatedby B cells and the antibodies they produce. Cell-mediated immunity iscontrolled by T cells. Both humoral and cell-mediated immunityparticipate in protecting the host from invading organisms. Thisinterplay can result in an effective killing or control of foreignorganisms. Occasionally, however, the interplay can become erratic. Inthese cases, there is a dysregulation that can cause disease. Sometimesthe disease is life-threatening, such as with septic shock and certainautoimmune disorders.

The B and T lymphocytes are critical components of a specific immuneresponse. B cells are activated by antigen to engender clones ofantigen-specific cells that mediate adaptive immunity. Most clonesdifferentiate to plasma cells that secrete antibody, while a few clonesform memory cells that revert to plasma cells. Upon subsequentre-infection, memory cells produce a higher level of antibody in ashorter period than in the primary response. Antibodies secreted by theplasma cells can play multiple roles in immunity, such as binding andneutralizing a foreign agent, acting as opsonins (IgG) to promotephagocytosis, directly affecting metabolism and growth of someorganisms, engaging in antigen-antibody reactions that activatecomplement, causing phagocytosis and membrane attack complex, and/orengaging in antigen-antibody reactions that activate T cells and otherkiller cells.

T lymphocytes function as both helper cells and suppressor cells. HelperT cells induce antigen-specific B cells and effector T cells toproliferate and differentiate. Suppressor T cells interact with helper Tcells to prevent an immune response or to suppress an ongoing one, or toregulate effector T cells. Cytotoxic T cells destroy antigen by bindingto target cells. In a delayed-type hypersensitivity reaction, the Tcells do not destroy antigen, but attract macrophages, neutrophils andother cells to destroy and dispose of the antigen.

T cells can detect the presence of intracellular pathogens becauseinfected cells display on their surface peptide fragments derived fromthe pathogens' proteins. These foreign peptides are delivered to thecell surface by specialized host-cell glycoproteins, termed MajorHistocompatibility Complex (MHC) molecules. The recognition of antigenas a small peptide fragment bound to a MHC molecule and displayed at thecell surface is one of the most distinctive features of T cells. Thereare two different classes of MHC molecules, known as MHC class I and MHCclass II, that deliver peptides from different cellular compartments tothe surface of the infected cell. Peptides from the cytosol are bound toMHC class I molecules which are expressed on the majority of nucleatedcells and are recognized by CD8+ T cells. MHC class II molecules, incontrast, traffic to lysosomes for sampling endocytosed protein antigenswhich are presented to the CD4+T cells (Bryant and Ploegh, Curr OpinImmunol 2004; 16:96-102).

CD8+ T cells differentiate into cytotoxic T cells, and kill the cell.CD4+ T cells differentiate into two types of effector T cells. Pathogensthat accumulate in large numbers inside macrophage vesicles tend tostimulate the differentiation of T_(H1) cells which activate macrophagesand induce B cells to make IgG antibodies that are effective inopsonizing extracellular pathogens for uptake by phagocytes.Extracellular antigens tend to stimulate the production of T_(H2) cellswhich initiate the humoral immune response by activating naiveantigen-specific B cells to produce IgM antibodies, inter alia.

The innate and adaptive immune systems interact, in that the cells ofthe innate immune system can express various molecules that can interactwith or trigger the adaptive immune system by activating certain cellscapable of producing immune factors, such as by activating T and B cellsof the lymphatic series of leukocytes. The early induced butnon-adaptive responses are important for two main reasons. First, theycan repel a pathogen or, more often, control it until an adaptive immuneresponse can be mounted. Second, these early responses influence theadaptive response in several ways. For example, the innate immuneresponse produces cytokines and other inflammatory mediators that haveprofound effects on subsequent events, including the recruitment of newphagocytic cells to local sites of infection. Another effect of thesemediators is to induce the expression of adhesion molecules on theendothelial cells of the local blood vessels, which bind to the surfaceof circulating monocytes and neutrophils and greatly increase their rateof migration of these cells out of the blood and into the tissues. Theseevents all are included under the term inflammation, which is a featureof the innate immune system that forms part of the protective responseat a localized site to isolate, destroy and remove a foreign material.This is followed by repair. Inflammation is divided into acute andchronic forms.

The immune system communicates via nonspecific tissue resistancefactors. These include the interferons, which are proteins produced inresponse to viruses, endotoxins and certain bacteria. Interferonsinhibit viral replication and activate certain host-defense responses.Infected cells produce interferon that binds the infected cells toother, neighboring cells, causing them to produce antiviral proteins andenzymes that interfere with viral gene transcription and proteinssynthesis. Interferons can also affect normal cell growth and suppresscell-mediated immunity.

Complement is another nonspecific tissue resistance factor, andcomprises plasma proteins and membrane proteins that mediate specificand non-specific defenses. Complement has two pathways, the classicalpathway associated with specific defense, and the alternative pathwaythat is activated in the absence of specific antibody, and is thusnon-specific. In the classical pathway, antigen-antibody complexes arerecognized when C1 interacts with the Fc of the antibody, such as IgMand to some extent, IgG, ultimately causing mast cells to releasechemotactic factors, vascular mediators and a respiratory burst inphagocytes, as one of many mechanisms. The key complement factorsinclude C3a and C5a, which cause mast cells to release chemotacticfactors such as histamine and serotonin that attract phagocytes,antibodies and complement, etc. Other key complement factors are C3b andC5b, which enhance phagocytosis of foreign cells, and C8 and C9, whichinduce lysis of foreign cells (membrane attack complex).

Gelderman et al. (Mol Immunol 2003; 40:13-23) reported thatmembrane-bound complement regulatory proteins (mCRP) inhibit complementactivation by an immunotherapeutic mAb in a syngeneic rat colorectalcancer model. While the use of mAb against tumor antigens and mCRPovercame an observed effect of mCRP on tumor cells, there has been nodirect evidence to support this approach. Still other attempts to usebispecific antibodies against CD55 and against a tumor antigen (G250 orEpCAM) have been suggested by Gelderman et al. (Lab Invest 2002;82:483-493; Eur J Immunol 2002; 32:128-135) based on in vitro studiesthat showed a 2-13-fold increase in C3 deposition compared to use of theparental antitumor antibody. However, no results involving enhanced cellkilling were reported. Jurianz et al. (Immunopharmacology 1999;42:209-218) also suggested that combining treatment of a tumor withanti-HER2 antibodies in vitro could be enhanced by prior treatment withantibody-neutralization of membrane-complement-regulatory protein, butagain no in vivo results were provided. Sier et al. (Int J Cancer 2004;109:900-908) recently reported that a bispecific antibody made againstan antigen expressed on renal cell carcinoma (Mab G250) and CD55enhanced killing of renal cancer cells in spheroids when beta-glucan wasadded, suggesting that the presence of CR3-priming beta-glucan wasobligatory.

Neutrophils, another cell involved in innate immune response, alsoingest, degrade and dispose of debris. Neutrophils have receptors forcomplement and antibody. By means of complement-receptor bridges andantibody, the foreign noxients can be captured and presented tophagocytes for engulfment and killing.

Macrophages are white blood cells that are part of the innate systemthat continually search for foreign antigenic substances. As part of theinnate immune response, macrophages engulf, kill and dispose of foreignparticles. However, they also process antigens for presentation to B andT cells, invoking humoral or cell-mediated immune responses.

The dendritic cell is one of the major means by which innate andadaptive immune systems communicate (Reis e Sousa, Curr Opin Immunol2004; 16:21-25). It is believed that these cells shape the adaptiveimmune response by the reactions to microbial molecules or signals.Dendritic cells capture, process and present antigens, thus activatingCD4+ and CD8+ naive T lymphocytes, leading to the induction of primaryimmune responses, and derive their stimulatory potency from expressionof MHC class I, MHC class II, and accessory molecules, such as CD40,CD54, CD80, CD86, and T-cell activating cytokines (Steinman, J ExpHematol 1996; 24:859-862; Banchereau and Steinman, Nature 1998;392:245-252). These properties have made dendritic cells candidates forimmunotherapy of cancers and infectious diseases (Nestle, Oncogene 2000;19:673-679; Fong and Engleman, Annu Rev Immunol 2000; 18:245-273;Lindquist and Pisa, Med Oncol 2002; 19:197-211), and have been shown toinduce antigen-specific cytotoxic T cells that result in strong immunityto viruses and tumors (Kono et al., Clin Cancer Res 2002; 8:394-40).

Also important for interaction of the innate and adaptive immune systemsis the NK cell, which appears as a lymphocyte but behaves like a part ofthe innate immune system. NK cells have been implicated in the killingof tumor cells as well as essential in the response to viral infections(Lanier, Curr Opin Immunol 2003; 15:308-314; Carayannopoulos andYokoyama, Curr Opin Immunol 2004; 16:26-33). Yet another importantmechanism of the innate immune system is the activation of cytokinemediators that alert other cells of the mammalian host to the presenceof infection, of which a key component is the transcription factor NF-κB(Li and Verna, Nat Rev Immunol 2002; 2:725-734).

As mentioned earlier, the immune system can overreact, resulting inallergies or autoimmune diseases. It can also be suppressed, absent, ordestroyed, resulting in disease and death. When the immune system cannotdistinguish between “self” and “nonself,” it can attach and destroycells and tissues of the body, producing autoimmune diseases, e.g.,juvenile diabetes, multiple sclerosis, myasthenia gravis, systemic lupuserythematosus, rheumatoid arthritis, and immune thrombocytopenicpurpura. Immunodeficiency disease results from the lack or failure ofone or more parts of the immune system, and makes the individualssusceptible to diseases that usually do not affect individuals with anormal immune system. Examples of immunodeficiency disease are severecombined immunodeficiency disease (SCID) and acquired immunodeficiencydisease (AIDS). The latter results from human immunodeficiency virus(HIV) and the former from enzyme or other inherited defects, such asadenosine deaminase deficiency.

Numerous and diverse methods of immunosuppression or of neutralizingproinflammatory cytokines have proven to be unsuccessful clinically inpatients with sepsis and septic shock anti-inflammatory strategies.(Riedmann, et al., cited above; Van Amersfoort et al. (Clin MicrobiolRev 2003; 16:379-414), such as general immunosuppression, use ofnonsteroidal anti-inflammatory drugs, TNF-α antibody (infliximab) or aTNF-R:Fc fusion protein (etanercept), IL-1 (interleukin-1) receptorantagonist, or high doses of corticosteroids. However, a success in thetreatment of sepsis in adults was the PROWESS study (Human ActivatedProtein C Worldwide Evaluation in Severe Sepsis (Bernard et al., N EnglJ Med 2001; 344:699-709)), showing a lower mortality (24.7%) than in theplacebo group (30.8%). This activated protein C (APC) agent probablyinhibits both thrombosis and inflammation, whereas fibrinolysis isfostered. Friggeri et al. (2012, Mol Med 18:825-33) reported that APCdegrades histones H3 and H4, which block uptake and clearance ofapoptotic cells by macrophages and thereby contribute to organ systemdysfunction and mortality in acute inflammatory states. Van Amersfoortet al. state, in their review (ibid.) that: “Although the blocking ormodulation of a number of other targets including complement andcoagulation factors, neutrophil adherence, and NO release, are promisingin animals, it remains to be determined whether these therapeuticapproaches will be effective in humans.” This is further emphasized in areview by Abraham, “Why immunomodulatory therapies have not worked insepsis” (Intensive Care Med 1999; 25:556-566). In general, although manyrodent models of inflammation and sepsis have shown encouraging resultswith diverse agents over the past decade or more, most agents translatedto the clinic failed to reproduce in humans what was observed in theseanimal models, so that there remains a need to provide new agents thatcan control the complex presentations and multiple-organ involvement ofvarious diseases involving sepsis, coagulopathy, and certainneurodegenerative conditions having inflammatory or immune dysregulatorycomponents.

More recent work on immunoglobulins in sepsis or septic shock has beenreported. For example, Toussaint and Gerlach (2012, Curr Infect Dis Rep14:522-29) summarized the use of ivIG as an adjunct therapy in sepsis.The metanalysis failed to show any strong correlation between generalimmunoglobulin therapy and outcome. LaRosa and Opal (2012, Curr InfectDis Rep 14:474-83) reported on new therapeutic agents of potential usein sepsis. Among other agents, anti-TNF antibodies are in currentclinical trials for sepsis, while complement antagonists have shownpromising results in preclinical models of sepsis. Nalesso et al. (2012,Curr Infect Dis Rep 14:462-73) suggested that combination therapies withmultiple agents may prove more effective for sepsis treatment. Theimmunopathogenesis of sepsis has been summarized by Cohen (2002, Nature420:885-91).

The immune system in sepsis is believed to have an early intenseproinflammatory response after infection or trauma, leading to organdamage, but it is also believed that the innate immune system oftenfails to effectively kill invading microorganisms (Riedmann and Ward,Expert Opin Biol Ther 2003; 3:339-350). There have been some studies ofmacrophage migration inhibitory factor (MIF) in connection with sepsisthat have shown some promise. For example, blockage of MIF or targeteddisruption of the MIF gene significantly improved survival in a model ofseptic shock in mice (Calandra et al., Nature Med 2000; 6:164-170), andseveral lines of evidence have pointed to MIF as a potential target fortherapeutic intervention in septic patients (Riedmann et al., citedabove). Bucala et al. (U.S. Pat. No. 6,645,493 B1) have claimed that ananti-MIF antibody can be effective therapeutically for treating acondition or disease caused by cytokine-mediated toxicity, includingdifferent forms of sepsis, inflammatory diseases, acute respiratorydisease syndrome, granulomatous diseases, chronic infections, transplantrejection, cachexia, asthma, viral infections, parasitic infections,malaria, and bacterial infections, which is incorporated herein in itsentirety, including references. The use of anti-LPS (lipopolysaccharide)antibodies alone similarly has had mixed results in the treatment ofpatients with septic shock (Astiz and Rackow, Lancet 1998;351:1501-1505; Van Amersfoort et al., Clin Microbiol Rev 2003;16:379-414.

Complement C5a, like C3a, is an anaphylatoxin. It mediates inflammationand is a chemotactic attractant for induction of neutrophilic release ofantimicrobial proteases and oxygen radicals. Therefore, C5a and itspredecessor C5 are particularly preferred targets. By targeting C5, notonly is C5a affected, but also C5b, which initiates assembly of themembrane-attack complex. Thus, C5 is another preferred target. C3b, andits predecessor C3, also are preferred targets, as both the classicaland alternate complement pathways depend upon C3b. Three proteins affectthe levels of this factor, C1 inhibitor, protein H and Factor I, andthese are also preferred targets according to the invention. Complementregulatory proteins, such as CD46, CD55, and CD59, may be targets towhich the multispecific antibodies bind.

Coagulation factors also are preferred targets according to theinvention, particularly tissue factor (TF), thrombomodulin, andthrombin. TF is also known also as tissue thromboplastin, CD142,coagulation factor III, or factor III. TF is an integral membranereceptor glycoprotein and a member of the cytokine receptor superfamily.The ligand binding extracellular domain of TF consists of two structuralmodules with features that are consistent with the classification of TFas a member of type-2 cytokine receptors. TF is involved in the bloodcoagulation protease cascade and initiates both the extrinsic andintrinsic blood coagulation cascades by forming high affinity complexesbetween the extracellular domain of TF and the circulating bloodcoagulation factors, serine proteases factor VII or factor VIIa. Theseenzymatically active complexes then activate factor IX and factor X,leading to thrombin generation and clot formation.

TF is expressed by various cell types, including monocytes, macrophagesand vascular endothelial cells, and is induced by IL-1, TNF-α orbacterial lipopolysaccharides. Protein kinase C is involved in cytokineactivation of endothelial cell TF expression. Induction of TF byendotoxin and cytokines is an important mechanism for initiation ofdisseminated intravascular coagulation seen in patients withGram-negative sepsis. TF also appears to be involved in a variety ofnon-hemostatic functions including inflammation, cancer, brain function,immune response, and tumor-associated angiogenesis. Thus, multispecificantibodies that target TF are useful not only in the treatment ofcoagulopathies, but also in the treatment of sepsis, cancer, pathologicangiogenesis, and other immune and inflammatory dysregulatory diseasesaccording to the invention. A complex interaction between thecoagulation pathway and the cytokine network is suggested by the abilityof several cytokines to influence TF expression in a variety of cellsand by the effects of ligand binding to the receptor. Ligand binding(factor VIIa) has been reported to give an intracellular calcium signal,thus indicating that TF is a true receptor.

Thrombin is the activated form of coagulation factor II (prothrombin);it converts fibrinogen to fibrin. Thrombin is a potent chemotaxin formacrophages, and can alter their production of cytokines and arachidonicacid metabolites. It is of particular importance in the coagulopathiesthat accompany sepsis. Numerous studies have documented the activationof the coagulation system either in septic patients or following LPSadministration in animal models. Despite more than thirty years ofresearch, the mechanisms of LPS-induced liver toxicity remain poorlyunderstood. It is now clear that they involve a complex and sequentialseries of interactions between cellular and humoral mediators. In thesame period of time, gram-negative systemic sepsis and its sequallaehave become a major health concern, attempts to use monoclonalantibodies directed against LPS or various inflammatory mediators haveyielded only therapeutic failures, as noted elsewhere herein.Multispecific antibodies according to the invention that target boththrombin and at least one other target address the clinical failures insepsis treatment.

A recombinant form of thrombomodulin has been approved for treatment ofdisseminated intravascular coagulation (DIC) and is in phase II clinicaltrials for DIC associated with sepsis (Okamoto et al., 2012, Crit CareRes Pract, Epub 2012 Feb. 28). Thrombomodulin has a pivotal role in theprotein C system that is important in the pathogensis of sepsis (Leviand Van der Poll, Minerva Anestesiol Epub Dec. 17, 2012). Downregulationof thrombomodulin in sepsis causes impaired activation of protein C thatis central in the modulation of coagulation and inflammation (Levi andVan der Poll, Minerva Anestesiol Epub Dec. 17, 2012). Administration ofrecombinant thrombomodulin is reported to have a beneficial effect onrestoration of coagulation and improvement of organ failure (Levi andVan der Poll, Minerva Anestesiol Epub Dec. 17, 2012). A recentretrospective study confirmed that treatment with recombinantthrombomodulin was associated with reduced mortality in hospitalizedpatients with sepsis-induced DIC (Yamakawa et al., 2013, Intensive CareMed, Epub Jan. 30, 2013).

In other embodiments, the multispecific antibodies bind to a MHC classI, MHC class II or accessory molecule, such as CD40, CD54, CD80 or CD86.The multispecific antibody also may bind to a T-cell activationcytokine, or to a cytokine mediator, such as NF-κB.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing glomerulonephritis in a patient. Exemplary kitsmay contain one or more histone-neutralizing agents, such as theanti-histone antibodies described herein. If the composition containingcomponents for administration is not formulated for delivery via thealimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used. In certain embodiments, atherapeutic agent may be provided in the form of a prefilled syringe orautoinjection pen containing a sterile, liquid formulation orlyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES Example 1 Effect of Histone-Neutralizing Agents on VascularNecrosis in Severe Glomerulonephritis

Severe glomerulonephritis involves cell necrosis as well as NETosis, aprogrammed neutrophil death leading to expulsion of nuclear chromatinleading to neutrophil extracellular traps (NETs). We speculated on arole of the dying cell's and NET's histone component in necrotizingglomerulonephritis. Histones from calf thymus or histones released byneutrophils undergoing NETosis killed glomerular endothelial cells,podocytes, and parietal epithelial cells in a dose-dependent manner. Asdiscussed below, this effect was prevented by histone-neutralizingagents such as anti-histone IgG, activated protein C, or heparin.

Histone toxicity on glomeruli ex vivo was TLR2/4-dependent. Lack ofTLR2/4 attenuated intra-arterial histone injection-induced renalthrombotic microangiopathy and glomerular necrosis in mice. Anti-GBMglomerulonephritis involved NET formation and vascular necrosis.Pre-emptive anti-histone IgG administration significantly reduced allaspects of glomerulonephritis, i.e. vascular necrosis, podocyte loss,albuminuria, cytokine induction, recruitment and activation ofglomerular leukocytes as well as glomerular crescent formation.

To evaluate the therapeutic potential of histone neutralization wetreated mice with established glomerulonephritis with three differenthistone-neutralizing agents. Anti-histone IgG, recombinant activatedprotein C, and heparin all abrogated severe glomerulonephritis,suggesting that histone-mediated glomerular pathology is not an initialbut rather a subsequent event in necrotizing glomerulonephritis.Together, histone release during glomerulonephritis elicits cytotoxicand immunostimulatory effects. Neutralizing extracellular histones istherapeutic in severe experimental glomerulonephritis.

Materials and Methods

Mice and anti-GBM nephritis model—C57BL/6 mice were procured fromCharles River (Sulzfeld, Germany), 6-8 week old mice received anintravenous injection of 100 μof anti-GBM serum (sheep anti-ratglomeruli basement membrane serum procured from Probetex INC, PTX-001).Urine samples were collected at different time points after antiseruminjection to evaluate the functional parameters of kidney damage. On day7 the mice were sacrificed by cervical dislocation to collect plasma andkidney tissue. Kidneys were kept at −80° C. for protein isolation and inRNALATER® solution at −20° C. for RNA isolation. A part of the kidneywas also kept in formalin to be embedded in paraffin for histologicalanalysis (Teixeira et al., 2005, Kidney Int 67:514B). We treated groupsof mice either with 20 mg/kg, i.p. control IgG or anti-histone antibody(clone BWA-3) to neutralize the effects of extracellular histones.

Assessment of renal pathology—Renal sections of 2 μm were stained withperiodic acid-Schiff reagent. Glomerular abnormalities were scored in 50glomeruli per section by a blinded observer. The following criteria wereassessed in each of the 50 glomeruli and scored as segmental or globallesions if less or more than 50% of the glomerular tuft were affected byfocal necrosis and capsule adhesions. Cellular crescents were assessedseparately when more than a single layer of PECs were present around theinner circumference of Bowman's capsule. Immunostaining was performed asdescribed using the following primary antibodies: for WT-1/nephrin,neutrophils (Serotec, Oxford, UK), Mac-2 (Cedarlane, Ontario, Canada),TNF-α (Abcam, Cambridge, UK) and fibrinogen (Abcam, Cambridge, UK).Stained glomerular cells were quantified in 50 glomeruli per section.

Electron microscopy—Kidney tissues and endothelial cell monolayers werefixed in 2.0% paraformaldehyde/2.0% glutaraldehyde, in 0.1M sodiumphosphate buffer, pH 7.4 for 24 h, followed by 3 washes×15 min in 0.1 msodium phosphate buffer, pH 7.4 and distilled water. For transmission EMkidneys were post-fixed, in phosphate cacodylate-buffered 2% OsO4 for 1h, dehydrated in graded ethanols with a final dehydration in propyleneoxide and embedded in Embed-812 (Electron Microscopy Sciences, Hatfield,Pa.). Ultrathin sections (˜90-nm thick) were stained with uranyl acetateand Venable's lead citrate. For scanning EM, after rinsing in distilledH₂O, cells on coverslips were treated with 1% thiocarbohydrazide,post-fixed with 0.1% osmium tetroxide, dehydrated in ethanol, mounted onstubs with silver paste and critical-point dried before being sputtercoated with gold/palladium. Specimens were viewed with a JEOL model1200EX electron microscope (JEOL, Tokyo, Japan).

Immunohistochemistry of human tissues—Formalin-fixed paraffin-embeddedsections of renal biopsies from five subjects with ANCA-positive RPGN,newly diagnosed in 2013, were drawn from the files of the Institute ofPathology at the Ludwig-Maximilians-University of Munich. The renalbiopsies were fixed in 4% PBS-buffered formalin solution and embedded inparaffin. Biopsies contained normal glomeruli and glomeruli exhibitingcellular, fibrocellular or fibrous crescents. Controls consisted ofnormal kidney tissue from tumor nephrectomies. TLR2 and TLR4 expressionwas assessed by using specific antibodies (TLR2-LS Bio, Seattle, Wash.,TLR4- Novus, Littleton, Co.).

In-Vitro Models

Cytotoxicity assay—Mouse glomerular endothelial cells (GEnC (46)),podocytes (47)), and parietal epithelial cells (PECs,(48)) were culturedin 96 well plates with RPMI media without FCS and PS and allowed toadhere overnight. The cells were stimulated with the differentconcentrations of total calf thymus histones (10, 20, 30, 40, 50 and 100μg/ml) with or without histone antibody for another 18-20 h.Cytotoxicity assay was performed using Promega CELLTITER 96®non-radioactive cell proliferation assay (MTT Assay Kit, Mannheim,Germany). Glomerular cells were also incubated with histones with orwithout anti-histone IgG, heparin and/or aPC. LDH assay usingcytotoxicity detection kit (Roche Diagnostics, Mannheim, Germany) wasused to assess cell death.

Podocyte detachment assay—Podocytes were grown at 33° C. using modifiedRPMI media in the presence of IFN-γ in collagen coated 10 cm dishes and8×10⁴ cells were seeded and allowed to differentiate as podocytes at 37°C. for two weeks in collagen plates without IFN-γ. Once the monolayersof podocytes were differentiated, the cells were treated with eitherhistones or GBM antiserum with or without histone antibody and allowedto sit for 18 h. Detached cells which are present in supernatant weremanually counted using an hemocytometer. Adhered cells were trypsinisedand counted manually to calculate the percentage of cells detached.

In-vitro tube formation assay—Matrigel was thawed overnight at 4° C. tomake it liquid. After 10 μl per well of μ-slide angiogenesis (IBIDI,Munich, Germany) was added, the gel was allowed to solidify at 37° C.GEnCs were seeded at 1×10⁴ cells/well and stimulated with VEGF and b-FGFas positive control or with histones with or without anti-histoneantibody. Tube formation as a marker of angiogenesis was assessed bylight microscopy by taking a series of pictures at 0 h, 4 h 8 h and 24 h(49).

NETosis assay—Neutrophils were isolated from healthy mice by dextransedimentation and hypotonic lysis of RBCs. Neutrophil extracellulartraps (NETs) were induced in-vitro by adding TNF-α (Immunotools,Friesoythe, Germany) or phorbol 12-myristate 13-acetate (PMA,Sigma-Aldrich, Mo., USA) for 12 h in with or without anti-histoneantibody. Endothelial cell death was assessed by MTT assay andimmunofluorescence staining for histones (BWA-3 clone), neutrophilelastase (ABCAM®, Cambridge, UK) and 4′,6-Diamidin-2-phenylindol (DAPI,Vector labs, Burlingame, Calif.) after fixing with acetone.

BMDCs and J774 macrophages—Bone marrow cells were isolated from healthymouse and plated at 1×10⁶ cells per well and differentiated into BMDCsin the presence of GM-CSF (Immunotools). J774 macrophage cells weregrown in RPMI media, plated at 1×10⁶ cells per well, and stimulated withdifferent doses of histones with or without anti-histone antibody for 18h. Supernatants were collected for TNF-α (Bio Legend, San Diego, Calif.)and IL-6 Elisa (BD Biosciences, San Diego, Calif.) determination. Flowcytometry for the activation markers MHC-II, CD40, CD103 and CD86 (BD)was also performed.

Flow cytometry—Flow cytometric analysis of cultured and renal immunecells was performed on a FACSCALIBUR™ flow cytometer (BD) as described(Lech et al., 2009, J Immunol 183:4109). Every isolate was incubatedwith binding buffer containing either anti-mouse CD11c, CD11b, CD103,F4/80, and CD45 antibodies (BD) for 45 min at 4° C. in the dark wereused to detect renal mononuclear phagocyte populations. Anti-CD86 (BD)was used as an activation marker. Anti-CD3 and CD4 (BD) were used toidentify the respective T-cell populations.

RNA preparation and real-time RT-PCR—Reverse transcription and real timeRT-PCR from total renal RNA was prepared as described (Patole et al.,2007, J Autoimmun 29:52). SYBR Green Dye detection system was used forquantitative real-time PCR on a Light Cycler 480 (Roche, Mannheim,Germany). Gene-specific primers (300 nM, Metabion, Martinsried, Germany)were used as follows: Reverse and forward primers respectively 18s:AGGGCCTCACTAAACCATCC (SEQ ID NO:111) and GCAATTATTCCCCATGAACG (SEQ IDNO:112), TNF-α: CCACCACGCTCTTCTGTCTAC (SEQ ID NO:113) andAGGGTCTGGGCCATAGAACT (SEQ ID NO:114), Fibrinogen (FGL-2):AGGGGTAACTCTGTAGGCCC (SEQ ID NO:115) and GAACACATGCAGTCACAGCC (SEQ IDNO:116), WT-1: CATCCCTCGTCTCCCATTTA (SEQ ID NO:117) andTATCCGAGTTGGGGAAATCA (SEQ ID NO:118), CD44: AGCGGCAGGTTACATTCAAA (SEQ IDNO:119) and CAAGTTTTGGTGGCACACAG (SEQ ID NO:120). Controls consisting ofddH₂O were negative for target and housekeeping genes.

Statistical Analysis

Data were expressed as mean±standard error of the mean (SEM). Comparisonbetween groups was performed by two-tailed t-test or ANOVA. A value ofp<0.05 was considered to be statistically significant. All statisticalanalyses were calculated using Graph Pad Prism (GraphPad).

Example 2 Glomerular TLR2 and TLR4 Expression in Severe HumanGlomerulonephritis

We first asked whether the TLR2 and TLR4 (Allam et al., 2012, J Am SocNephrol 23:1375) extracellular histones were expressed in the healthyand diseased glomeruli. TLR2/4 immunostaining of normal human kidneyshowed a weak granular positivity in all glomerular cells. TLR4positivity was clearly noted in glomerular endothelial cells (FIG. 1A).In addition, TLR2 was strongly positive in the cytoplasm of epithelialcells of the proximal and distal tubule, while this was less prominentfor TLR4 (FIG. 1A). Immunostaining of kidney biopsies of patients withANCA-associated necrotizing and crescentic GN revealed prominentpositivity also in PECs along the inner aspect of Bowman's capsule (FIG.1B). As glomerular crescents are largely formed by PECs (Smeets et al.,2009, J Am Soc Nephrol 20:2593; Smeets et al., 2009, J Am Soc Nephrol20:2604), glomerular crescents displayed TLR2 and TLR4 positivity (FIG.1C). Thus, the cells of the normal glomerulus express TLR2/4 and PECsinduce these TLRs in crescentic GN.

Example 3 Anti-histone IgG Prevents Histone Toxicity on Glomerular Cells

Histones were previously shown to be toxic to pulmonary endothelialcells in vitro and in vivo (Xu et al., 2009, Nat Med 15:1318; Abrams etal., 2013, Am J Respir Crit Care Med 187:160). We tested this effect oncultured glomerular endothelial cells and found that a total histonepreparation was cytotoxic in a dose-dependent manner. Anti-histone IgGderived from the BWA-3 hybridoma is known to neutralize the toxic andimmunostimulatory effect of extracellular histones (Xu et al., 2009, NatMed 15:1318; Xu et al., 2011, J Immunol 187:2626; Monestier et al.,1993, Mol Immunol 30:1069. Anti-histone IgG almost entirely preventedhistone toxicity on glomerular endothelial cells up to a histoneconcentration of 30 μg/ml (FIG. 2A). Anti-histone IgG also preventedhistone-induced GEnC microtubule destruction in angiogenesis assays(FIG. 9A-9B). Histone-induced toxicity was also evident in culturedpodocytes and PECs albeit at much higher histone concentrations comparedto the toxic dose required to kill endothelial cells (FIG. 2A).Anti-histone-IgG also significantly reduced histone-induced detachmentof cultured podocytes (FIG. 10). Thus, extracellular histones are toxicto glomerular cells, which toxicity can be blocked by anti-histone IgG.

Example 4 Neutrophil Extracellular Traps Kill Glomerular EndothelialCells through Histone Release

In severe GN neutrophils undergo NETosis, which deposits nuclearchromatin within the glomerular capillaries (Kessenbrock et al., 2009,Nat Med 15:623). Immunohistochemical staining showed nuclear chromatinrelease from netting neutrophils, including the spread of histonesoutside the dying cells (FIG. 2B). Neutrophils undergoing TNF-α- orPMA-induced NETosis on monolayers of glomerular endothelial cellsdestroyed this monolayer by inducing endothelial cell death, while TNFor PMA alone did not (FIG. 2C-2E). This NETosis-related endothelial celltoxicity was entirely prevented by anti-histone IgG (FIG. 2C-2E). Weconclude that netting neutrophils damage glomerular endothelial cellsvia the release of histones.

Example 5 Histones Need TLR2/4 to Trigger Glomerular Necrosis andMicroangiopathy

Whether glomerular toxicity of extracellular histones isTLR2/4-dependent is not clear. To answer this question we exposedglomeruli isolated from wild type and Tlr2/4-deficient mice to histonesex vivo. Histones exposure was cytotoxic to glomeruli, a process thatwas entirely prevented using glomeruli from Tlr2/4-deficient mice (FIG.3A). Lack of TLR2/4 also prevented IL-6 and TNF expression inhistone-exposed glomeruli (FIG. 11). We also studied the effects ofextracellular histones on glomeruli in vivo. Because intravenous histoneinjection kills mice immediately by pulmonary microvascular injury (Xuet al., 2009, Nat Med 15:1318), we injected histones directly into theleft renal artery in anaesthetized mice. Unilateral histone injectioncaused glomerular lesions within 24 hours ranging from minor endothelialfibrinogen positivity to thrombotic microangiopathy and globalglomerular necrosis (FIG. 3B-3C). The contralateral kidney remainedunaffected (not shown). Histone injection into the renal artery ofTlr2/4-deficient mice showed significantly reduced glomerular lesionsand fibrinogen positivity (FIG. 3B). These results demonstrate thatextracellular histones induce glomerular injury in a TLR2/4-dependentmanner.

Example 6 Extracellular Histones Contribute to Severe Glomerulonephritis

Based on these results we speculated that intrinsic histone release mayalso contribute to severe GN in vivo. To address this question weapplied the same neutralizing anti-histone IgG as used in vitro thatdemonstrated the functional contribution of extracellular histones inlethal endotoxemia (Xu et al., 2009, Nat Med 15:1318). Mice wereinjected i.p. with 20 mg/kg anti-histone IgG or with 20 mg/kg controlIgG 24 hours before the intravenous injection of a GBM antiserum raisedin sheep. At the end of the study at day 7 only sheep IgG but no mouseIgG deposits were found in glomeruli, excluding any autologousanti-sheep IgG response contributing to glomerulonephritis (FIG. 12A).Anti-histone IgG significantly reduced blood urea nitrogen (BUN) andserum creatinine levels following GBM antiserum injections (FIG. 4A).This was associated with a significant reduction in crescent formationand global glomerular pathology with less severe lesions 7 days afterantiserum injection (FIG. 4B-4C). Myeloperoxidase (MPO) immunostainingvisualized NETs inside glomeruli, which was associated with focal lossof endothelial CD31 positivity as a marker of glomerular vascular injury(FIG. 4D). Anti-histone IgG did not affect extracellular positivity butmaintained CD31+ vasculature (FIG. 4D), indicating a protective effecton NET-related vascular injury.

Because histones were toxic to glomerular endothelial cells andpodocytes in vitro, we assessed the glomerular capillary ultrastructureby transmission electron microscopy. In control mice with crescenticglomeruli there was severe glomerular damage with fibrin depositsreplacing large glomerular segments (fibrinoid necrosis). The capillaryloops showed extensive GBM splitting and thinning, prominent endothelialcell nuclei, massive subendothelial edema with closure of theendothelial fenestrae, and obliteration of the capillary lumina.Subendothelial transudates (leaked serum proteins) and luminal plateletsand neutrophils were also noted. Severe podocyte injury with diffusefoot process effacement, reactive cytoplasmic changes and detachmentfrom the GBM were apparent (FIG. 5A).

In contrast, glomeruli of mice injected with anti-histone IgG showedrestored endothelial fenestrations, flat appearing endothelial cells andpreserved podocytes with intact foot processes (FIG. 5A). WT-1/nephrinco-immunostaining revealed that anti-histone IgG largely preventedpodocyte loss in antiserum-induced GN (FIG. 5B-5C). This was consistentwith significant reduction of albuminuria on day 7 following antiseruminjection as compared to control IgG-treated mice (FIG. 5D). Theseresults demonstrate that extracellular histones induce severe GN bycausing glomerular vascular injury and podocyte loss, accompanied byproteinuria. They also demonstrate the efficacy of anti-histone antibodyin preventing glomerular damage in glomerulonephritis.

Example 7 Extracellular Histones Drive Glomerular Leukocyte Recruitmentand Activation

Infiltrating leukocytes are not only a documented source ofextracellular histones in severe GN (Kessenbrock et al., 2009, Nat Med15:623) but also important effector cells (Kurts et al., 2013, Nat RevImmunol 13:738). For example, in GBM antiserum-exposed glomerularendothelial cells, histone exposure triggered CXCL2 expression (FIG.12B). In vivo, anti-histone IgG significantly reduced the numbers ofglomerular neutrophils and macrophages as quantified by immunostaining(FIG. 6A). Flow cytometry of renal cell suspensions allowed us to betterdistinguish renal mononuclear phagocyte populations. Anti-histone IgGsignificantly reduced the numbers of activated (MHC II+) F4/80+ cells aswell as of activated (CD86+) CD11b/CD103+ cells, and of CD4+ dendriticcells (FIG. 6B). In fact, histones dose-dependently induced activationmarkers like MHCII, CD40, CD80, and CD86 also in cultured bone marrowderived macrophages (BMDCs), which was entirely prevented withanti-histone IgG (FIG. 6C). Taken together, extracellular histonestrigger glomerular leukocyte recruitment and activation, which can beblocked with anti-histone IgG in vitro and in vivo.

Example 8 Extracellular Histones Trigger Intraglomerular TNF-α Releaseand Thrombosis

Activated mononuclear phagocytes are also an important source ofpro-inflammatory cytokines in glomerular disease. Among these, TNF-αparticularly contributes to podocyte loss, proteinuria, andglomerulosclerosis (Ryu et al., 2012, J Pathol 226:120). Becauseanti-histone IgG entirely prevented histone-induced TNF-α secretion incultured macrophages and dendritic cells (FIG. 7A), we next assessedglomerular TNF-α expression. Immunostaining displayed robust TNF-αpositivity within the glomerular tuft, which not only localized ininfiltrating cells but also in inner and outer aspect of the glomerularcapillaries (FIG. 7B). Anti-histone IgG strongly reduced glomerularTNF-α positivity, which was consistent with the corresponding renal mRNAexpression levels (FIG. 7C). TNF-α is not only an inducer of NETosis butalso triggers a prothrombotic activation of (glomerular) endothelialcells and intravascular fibrin formation (32-34). Our GN model displayedglobal fibrinogen positivity within glomerular capillaries, which wasalmost entirely prevented with anti-histone IgG (FIG. 7D). Alsofibrinogen mRNA levels were reduced in the anti-histone IgG group (FIG.7E). These results show that extracellular histones triggerintraglomerular TNF-α production and microthrombi formation withinglomerular capillaries.

Example 9 Extracellular Histones Activate Parietal Epithelial Cells viaTLR2/4

Mitogenic plasma proteins leaking from injured glomerular capillariescause PEC hyperplasia and glomerular crescent formation (Ryu et al.,2012, J Pathol 228:482; Smeets et al., 2009, J Am Soc Nephrol 20:2593;Smeets et al., 2009, J Am Soc Nephrol 20:2604). In fact, inantiserum-induced GN glomerular crescents were positive forclaudin-1/WT-1 positive cells (FIG. 7F), where claudin-1 identifies PECsand WT-1 marks PEC activation (Shankland et al., 2013, Curr Opin NephrolHypertens 22:302. PECs cultured in 10% serum started proliferating uponhistone exposure (FIG. 7G). Having shown that TLR2 and -4 areupregulated in PECs during severe human GN, we questioned whetherextracellular histones drive PEC activation in a TLR2/4-dependentmanner.

The mitogenic effect of histones to serum exposed PECs was entirelyblocked by TLR2/4 inhibition (FIG. 7H). TLR2/4 inhibition also blockedhistone-induced expression of CD44 and WT-1 in PECs (FIG. 7I). Previousreports documented that heparin and recombinant activated protein C(aPC) also block histone toxicity (Xu et al., 2009, Nat Med 15:1318;Wildhagen et al., 2013, Blood 123:1098). As such, the protective effecton PEC activation was shared by anti-histone IgG, heparin or activatedprotein C (aPC) (FIG. 7I), the latter two suppressing histonecytotoxicity on glomerular endothelial cells just like anti-histone IgG(not shown). Thus, extracellular histones activate PECs in aTLR2/4-dependent manner, a process that may act synergistically withother triggers of PEC hyperplasia during crescent formation and that canbe blocked by anti-histone IgG, aPC or heparin.

Example 10 Delayed Onset of Histone Neutralization Still Improves SevereGN

The results of pre-emptive histone neutralization proved theirpathogenic contribution to severe GN. We mext examined whether histoneneutralization could be therapeutic in established disease. Anti-histoneIgG, heparin, and aPC all completely blocked histone toxicity onglomeruli ex vivo (FIG. 8A). In another series of experiments weinitiated anti-histone IgG, heparin, and aPC treatments 24 hours afterGBM antiserum injection, a time point where massive proteinuria andelevated BUN were already established (FIG. 4A, FIG. 5D). All thesetreatments consistently and significantly reduced plasma creatininelevels, proteinuria, and podocyte loss at day 7 (FIG. 8B-D). Histoneblockade also significantly reduced the percentage of glomeruli withglobal lesions or halted damage (FIG. 8E). Glomerular crescents werereduced by 80% (FIG. 8F) and so were features of secondary tubularinjury (FIG. 8G). This was associated with less glomerular neutrophiland macrophage infiltrates as well as a significant reduction ofintrarenal leukocyte subpopulations as well as their activation, asidentified by flow cytometry (FIG. 14). Thus, delayed onset of histoneblockade with anti-histone IgG, heparin or aPC protects from renaldysfunction and structural injury during severe GN.

Example 10 Summary of Effects of Histone Neutralization on Necrosis inGlomerulonephritis

We hypothesized that extracellular histones elicit toxic andimmunostimulatory effects on glomerular cells during necrotizing andcrescentic GN. The data reported in the Examples above confirm thisconcept and also demonstrate that histone neutralization continues to beprotective when it commences after disease onset, which implies apotential therapeutic use of histone neutralizing agents in severe GN.

Necrotizing and crescentic GN, such as seen in ANCA-associated renalvasculitis or anti-GBM disease, is associated with neutrophil-inducedglomerular injury. First discovered in 2004, NETosis is a regulated formof neutrophil death that supports killing of extracellular bacteria(Brinkmann et al., 2004, Science 303:1532). NETosis is not limited toantibacterial host defence but also occurs in sterile forms ofinflammation, because it can be triggered by pro-inflammatory cytokinessuch as TNF-α. Our in vitro studies show that TNF-α is a sufficientstimulus to trigger NETosis-driven injury of glomerular endothelialcells. NETosis releases many aggressive proteases, oxygen radicals, andpotential DAMPs into the extracellular space that have the potential todrive vascular injury in the glomerulus.

Our data demonstrate an essential role of histones in this context. Theendothelial toxicity of extracellular histones was first described in aseminal paper on sepsis, where early lethality was due to microvascularendothelial cell injury in the lung (Xu et al., 2009, Nat Med 15:1318).Subsequent reports further explored the thrombogenic potential ofextracellular histones via direct activation of endothelial cells aswell as of platelets (Abrams et al., 2013, Am J Respir Crit Care Med187:160; Saffarzadeh et al., 2012, PLoS One 7:e32366; Semeraro et al.,2011, Blood 118:1952; Ammollo et al., 2011, J Thromb Haemost 9:1795;Fuchs et al., 2011, Blood 118:3708; Fuchs et al., 2010. Proc Natl AcadSci USA 107:15880).

In infection and sepsis models, NETosis is the most likely source ofextracellular histones. However, in mechanical trauma, toxic liverinjury, cerebral stroke, and post-ischemic renal tubular necrosishistones are also released from dying tissue cells (Allam et al., 2014,J Mol Med, 92:465; Allam et al., 2012, J Am Soc Nephrol 23:1375). Thesource of extracellular histones in our in vivo model could be dyingglomerular cells as well as netting neutrophils, which we identified byMPO staining in situ. Histone blockade had no effect on NETosis per sebut rather worked on the related vascular injury inside the glomerulus.

Our in vitro and in vivo data clearly demonstrate that extracellularhistones are toxic to glomerular cells and promote glomerular injury inhealthy mice upon intra-arterial injection or during severeantiserum-induced GN. The mechanisms of histone toxicity are notentirely clear but are thought to be due to their strong basic charge(Gillrie et al., 2012, Am J Pathol 180:1028). While histones basiccharge is needed inside the nucleus to neutralize acidic residues of theDNA, outside the cell, it has the capacity to damage cell membranes(Gillrie et al., 2012, Am J Pathol 180:1028). The polyanion heparinblocks this charge effect of histones, which may explain itsantagonistic effect on histone toxicity in vitro and in vivo. However,we and others discovered that histones elicit also DAMP-like activity byactivating TLR2, TLR4, and NLRP3 (Semeraro et al., 2011, Blood 118:1952;Allam et al., 2012, J Am Soc Nephrol 23:1375; Allam et al., 2013, Eur JImmunol 43:3336; Huang et al., 2013, J Immunol 191:2665; Xu et al.,2011, J Immunol 187:2626), which is another pathway of how extracellularhistones trigger sterile inflammation.

Because TLR2 and TLR4 (but not NLRP3) are known to induce glomerularinjury in the heterologous anti-GBM GN model (Brown et al., 2006, JImmunol 177:1925; Brown et al., 2007, J Am Soc Nephrol 18:1732;Lichtnekert et al., 2011, PLoS One 6:e26778; Lichtnekert et al., 2009,Am J Physiol Renal Physiol 296:F867), we further explored thehistone-TLR2/4 axis. Tlr2/4-deficient glomeruli were protected fromhistone-induced injury ex vivo and in vivo, implying that thehistone-related glomerular injury relates to the TLR2/4-dependent DAMPeffect. In particular the presence of serum turned the cytotoxic effectof histones on PECs into PEC proliferation, which was entirely TLR2/4dependent. Although PEC necrosis can be followed by excessive PECrecovery leading to PEC hyperplasia and crescent formation (Sicking etal., 2012, J Am Soc Nephrol 23:629), concomitant plasma leakage andhistone release provide additional mitogenic stimuli during severe GN(Ryu et al., 2012, J Pathol 228:382).

Our proof-of-concept experiments were based on pre-emptive histoneneutralization with anti-histone IgG. To explore a potential efficacy ofhistone blockade in severe GN we also applied three different modes ofhistone inactivation following GN induction. Delayed onset ofanti-histone IgG was equally protective as pre-emptive therapy in termsof glomerular injury, proteinuria, and serum creatinine levels. The sameapplies to heparin treatment, which confirms previously publishedresults in GN models (Floege et al., 1993, Kidney Int 43:369). Our dataclearly show that heparin inhibits the direct toxic effects of histoneson glomerular endothelial cells, which is consistent with the results ofother investigators in other cell types (Hirsch, 1958, J Exp Med108:925; Ammollo et al., J Thromb Haemost 9:1795; Fuchs et al., 2010,Proc Natl Acad Sci USA 107:15880). As previously reported aPC degradesextracellular histones (Xu et al., 2009, Nat Med 15:1318. In the currentstudies it was equally effective as anti-histone IgG and heparin inabrogating extracellular histone toxicity in vitro and severe GN invivo.

Together, NETosis releases histones into the extracellular space wherethey have toxic effects on glomerular endothelial cells and podocytes.Extracellular histone-induced glomerular injury is partially due toTLR2/4. Pre-emptive as well as delayed onset of histone neutralizationeither by anti-histone IgG, recombinant aPC or heparin abrogates allaspects of GBM antiserum-induced severe GN. We conclude thatextracellular histones represent a novel therapeutic target in severeGN.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the invention.

What is claimed is:
 1. A method of treating glomerulonephritiscomprising administering to a subject with glomerulonephritis (GN) atleast one anti-histone agent selected from the group consisting of ananti-histone antibody or antigen-binding fragment thereof, activatedprotein C (APC), and heparin.
 2. The method of claim 1, wherein theanti-histone antibody binds to a human histone selected from the groupconsisting of histone H1/H5, histone H2A, histone H2B, histone H3 andhistone H4.
 3. The method of claim 1, wherein the anti-histone antibodyis an anti-histone H4 antibody.
 4. The method of claim 1, wherein theanti-histone antibody is selected from the group consisting of BWA-3,LG2-1 and LG2-2.
 5. The method of claim 1, wherein theglomerulonephritis is rapidly-progressive glomerulonephritis (RPGN). 6.The method of claim 1, wherein the glomerulonephritis is anti-neutrophilcytoplasmic antibody (ANCA) associated glomerulonephritis.
 7. The methodof claim 1, wherein the anti-histone agent inhibits activity oftoll-like receptor 2 (TLR-2) and TLR-4.
 8. The method of claim 1,wherein administration of anti-histone antibody reduces vascularnecrosis, podocyte loss, albuminuria, cytokine induction, recruitmentand activation of glomerular leukocytes and glomerular crescentformation.
 9. The method of claim 1, wherein the anti-histone antibodyis a chimeric, humanized or human antibody.
 10. The method of claim 1,wherein the antibody fragment is selected from the group consisting ofF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and single domain antibody(nanobody).
 11. The method of claim 1, further comprising administeringtwo or more anti-histone agents to the subject.
 12. The method of claim11, wherein the two or more agents are selected from the groupconsisting of an anti-histone antibody, activated protein C and heparin.13. The method of claim 1, wherein the subject is a human subject. 14.The method of claim 1, wherein administration of the anti-histone agentis effective to treat vascular necrosis in severe glomerulonephritis.15. The method of claim 1, further comprising administering to thesubject an anti-TNF-α antibody.
 16. The method of claim 1, wherein thesubject is a human subject.
 17. The method of claim 1, furthercomprising administering to the subject an antibody against toll-likereceptor 2 (TLR-2) or TLR-4.
 18. The method of claim 1, wherein theanti-histone antibody or antigen-binding fragment thereof is notconjugated to a therapeutic agent.
 19. The method of claim 1, whereinthe anti-histone antibody or antigen-binding fragment thereof isconjugated to at least one therapeutic agent.
 20. The method of claim19, wherein the therapeutic agent is selected from the group consistingof a second antibody, an antigen-binding fragment of a second antibody,a radionuclide, an immunomodulator, an anti-angiogenic agent, apro-apoptotic agent, a cytokine, a chemokine, a drug, a toxin, ahormone, an siRNA and an enzyme.
 21. The method of claim 20, furthercomprising administering to the subject an immunomodulator selected fromthe group consisting of a cytokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interleukin (IL), erythropoietin, thrombopoietin, tumor necrosisfactor (TNF), granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, interferon-λ, TGF-α, TGF-β, interleukin-1(IL-1), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23,IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin andlymphotoxin.
 22. The method of claim 21, wherein the cytokine isselected from the group consisting of human growth hormone, N-methionylhuman growth hormone, bovine growth hormone, parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein,tumor necrosis factor-α, tumor necrosis factor-β, mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, thrombopoietin (TPO),NGF-β, platelet-growth factor, TGF-α, TGF-β, insulin-like growthfactor-I, insulin-like growth factor-II, erythropoietin (EPO),osteoinductive factors, interferon-α, interferon-β, interferon-γ,macrophage-CSF (M-CSF), IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin,endostatin, tumor necrosis factor and lymphotoxin.
 23. The method ofclaim 1, further comprising administering to the subject a secondantibody or antigen-binding fragment thereof, where the second antibodybinds to an antigen selected from the group consisting of histone H2B,histone H3, histone H4, a proinflammatory effector of the innate immunesystem, a proinflammatory effector cytokine, a proinflammatory effectorchemokine, TNF-α, MIF, CD74, HLA-DR, IL-1, IL-3, IL-4, IL-5, IL-6, IL-8,IL-12, IL-15, IL-17, IL-18, IL-23, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,IL-18R, CD40L, CD44, CD46, CD55, CD59, CCL19, CCL21, mCRP, MCP-19,MIP-1A, MIP-1B, RANTES, ENA-78, IP-10, GRO-β, lipopolysaccharide,lymphotoxin, HMGB-1, tissue factor, a complement regulatory protein, acoagulation factor, thrombin, a complement factor, C3, C3a, C3b, C4a,C4b, C5, C5a, C5b, Flt-1 and VEGF.
 24. The method of claim 1, whereinthe antibody is a bispecific antibody comprising a first binding sitefor a histone and a second binding site for a non-histone antigen. 25.The method of claim 1, wherein the anti-histone antibody orantigen-binding fragment thereof is a fusion protein.